ITPI990038A1 - CYANOBACTERIAL STOCK OF THE NOSTOC GENUS, AS WELL AS CULTURES, BIOMASSES, EXTRACTS, BIOACTIVE MOLOCULES DERIVED FROM SUCH STOCK AND THEIR USE IN - Google Patents

Description

DESCRIPTION

Scope of the invention

The present invention relates to a new cyanobacterial strain of the genus Nostoc.

Furthermore, the invention relates to crops, biomasses, extracts and bioactive molecules derived from this strain and their use in the agricultural and pharmaceutical fields.

In particular, but not exclusively, the invention refers to the use of said cultures, biomasses, extracts and bioactive molecules as antifungals and antitumors.

The invention can be framed as a result of research in the field of antifungal, antitumor, antibiotic substances in the broad sense extractable from plant microorganisms.

In particular, in the agricultural field there is an increasing need to identify new substances capable of inhibiting the growth of fungal pathogens, which cause significant damage to crops.

Likewise, in the pharmacological field, the search for anticancer agents capable of inhibiting the growth of cancer cells is continuing.

Summary of the invention

The present invention, therefore, provides a new cyanobacterial strain of the genus Nostoc, usable in the agricultural and pharmaceutical fields.

A culture of the microorganism according to the invention, named KONA97, has been deposited under the Budapest Treaty and is part of the microorganism collection of the ATCC, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, where it is available under the ATCC access number

Still according to the invention, cultures, biomasses, relative extracts obtained from the aforementioned KONA97 strain are supplied, as well as bioactive molecules present in such crops, biomasses and extracts.

In particular, bioactive molecules with molecular weight 712 present in these cultures, biomasses and extracts have shown particular antifungal and antitumor activity.

Another aspect of the invention is the use in the agricultural and pharmaceutical fields of crops, biomass, extracts and bioactive molecules resulting from the KONA97 cyanobacterial strain.

In particular, the lipophilic extract of KONA97 was found to completely inhibit the growth of Colletotrichum tri folli, Penicillium expansum and Rhizoctonia solani, while minor degrees of inhibition were observed against Candida albicans, Phoma sp. and Fusarium roseum.

The lipophilic extract proved lethal for both Artemia salina and Brachionus sp. and inhibited the growth of HCT8 tumor line cells in proportion to the concentrations tested.

Obviously, cyanobacterial strains resulting from mutation, variation or recombination of the KONA97 strain mentioned above are also object of the invention, as well as bioactive molecules obtained by isolation from microorganisms or synthetically having the same characteristics as those derived from KONA97.

Further aspects of the cyanobacterium according to the invention will become clearer with the following description of its isolation, of its characteristics and properties, of the biomass production method and of the extraction techniques of the active principles derived therefrom.

During the description, reference will be made to the attached tables and graphs in which:

- table 1 indicates the composition of a mineral medium used for the cultivation of the KONA97 strain; - table 2 summarizes an elution method used to obtain the first accumulation of active fraction in the chemical characterization process of the active molecules of the extracts;

- table 3 summarizes an elution method used for the analytical separation of the isolated fraction with a semi-preparative column;

- figure 1 is a graphical representation of the elution method in table 3, and reports the percentage of acetonitrile in H20 / H + in ordinates and time in the abscissas. The gradient in the sigmoidal curve is obtained by coupling two quadratic curves together (via software).

Detailed description of the invention

a) Isolation of the microorganism

The Nostoc sp. KONA97 was isolated from a lava rock sample from the island of Kona (Hawaii), using the procedure described below. Small rock fragments were seeded in a flask containing 100 ml of BG-110 mineral culture medium modified with the addition of NaHC03 (composition in table 1), added with 200 mg l <"1> of cycloheximide for the control of chlorophytes and fungi.

The enrichment culture was then incubated at a temperature of 25 ° C under continuous lighting conditions (20 μιηοΐΐ photons m <'2> s <' 1>). After about 40 days the development of small green colonies adhering to the bottom of the flask was observed. The colonies, upon observation under the microscope, were found to be formed by cyanobacterial filaments whose morphological characteristics were attributable to those of the genus Nostoc.

Subsequently, the organism was isolated by seeding in Petri dishes on agarized BG-110 culture medium. For this purpose, a small aliquot of the enrichment culture was sown, after suitable dilutions, on the surface of the agar medium. The plates were placed to develop under the conditions previously described until the appearance of the cyanobacterial colonies.

a strain of Nostoc, called Nostoc sp. KONA97.

The purification of the culture from contaminating bacteria was carried out by exploiting the ability of cyanobacteria of the genus Nostoc to form acinetes (structures resistant to high temperatures) in the stationary phase of growth.

A crop of Nostoc sp. KONA97 with acineti was treated for 10 minutes at a temperature of 55 ° C and then diluted and sown on agar medium. By direct observation of the plates under the microscope at 120 magnification, the colonies formed on the surface of the medium that were apparently free from bacterial contaminants were selected and isolated.

Nostoc cultures obtained from isolations were tested for microbiological purity by sowing them in test tubes containing BG-110 medium enriched with glucose (5 g l "1) and casaminic acids (2 g l" 1). The cultures were incubated in the dark at 30 ° C for 15 days and the absence of bacteria was ascertained both macroscopically due to the absence of turbidity and by observation under a phase contrast microscope.

b) Description of the Nostoc KONA97 strain Morphological characteristics

Under microscopic observation, the organism is made up of long unbranched filaments with a supple or twisted course and surrounded by a hyaline capsule. The mature filaments are formed by cells in a chain, barrel-shaped, with a diameter of 5.2-6.5 μπι and have intercalary heterocysts or, more rarely, spherical-shaped terminals with a diameter of 5.6-6.8 μτη . The acinetes, spherical in shape with a diameter of 8-10.2 μηι, with a granular content, are formed in short chains distal to the heterocysts. Hormogones are mobile and formed by cylindrical cells with a diameter of about 3.5 μηι and a height equal to the diameter or slightly greater. The multiplication occurs both by formation of hormogones, both by fragmentation of the filaments, preferably at the level of the acineti, and by germination of the latter.

Cultural characteristics

Colonies on solid medium are raised, with jagged edges and a slightly wavy and shiny surface. The colonies are formed by long heterocystate filaments with a flexuous to twisted course, joined by a thickening mucilaginous matrix. The color is bright green.

In liquid culture, the organism develops in small flakes uniformly dispersed in the middle, of an intense green color.

Property

The organism is photoautotrophic and facultative chemoheterotrophic. In the dark it grows slowly on sucrose, glucose and fructose.

c) Biomass production

The biomass was produced starting from an inoculum represented by the maintenance culture of the cyanobacterium in liquid or agar medium. The culture was carried out in "batch" using a multistep procedure, incubating the cultures in a thermostat equipped with a stirring system, at a temperature of 30 ° C, under continuous lighting conditions (80 μτηοϋ photons m <"2> s <1> ) and in an air-carbon dioxide atmosphere (95/5, v / v) All the phases of the culture as well as the transfers were carried out under sterile conditions.

Once the cell concentration of about 1.5 g I <'1> was reached, the biomass was collected by filtration on nylon cloth with pores of about 12 µm and frozen at -20 ° C. d) Extraction

The extraction process was carried out starting from the freeze-dried biomass and ground in a mortar until it was reduced to a fine powder.

Two extraction methods were used: one in the screening phase to determine the spectrum of biological activity, the other in the subsequent chemical characterization phase of the extract.

Screening phase

In this phase, the cyanobacterial biomass was subjected to two successive extractions which led to the obtaining of a hydrophilic and a lipophilic extract.

To each gram of lyophilized biomass prepared as described above, 40 ml of an ethanol / water mixture (3: 7) was added and the resulting suspension was left to stand at 4 ° C overnight. Subsequently, the suspension was centrifuged at 10000 g for 10 minutes at 10 ° C and the supernatant was dried in Rotavapor at a temperature below 40 ° C. The hydrophilic extract was obtained by taking up the dry residue in a small volume of water.

A 1: 1 mixture of isopropanol and methylene chloride was added to the pellet obtained by centrifugation in the proportions of 40 ml of mixture per gram of lyophilized starting biomass. The suspension was left to stand overnight at 4 ° C and then filtered on creased paper. A solution was thus obtained which was subsequently evaporated in the Rotavapor at a temperature below 40 ° C. The residue, green in color due to the content of fat-soluble pigments, resuspended in a minimum volume of ethanol, represented the lipophilic extract.

Characterization phase

In this phase, the method for obtaining extracts to be used in the isolation and chemical characterization phase of the active ingredient was developed.

100 ml of methanol were added to each gram of biomass and the suspension obtained was stirred overnight. The suspension was then filtered on folded paper and 50 ml of water per gram of lyophilized starting biomass were added to the solution thus obtained. On the methanol / water mixture, transferred to a separator funnel, an extraction was carried out with 100 ml of methylene chloride per gram of lyophilized starting biomass. It has been ascertained that it is preferable to carry out two successive extractions, using half the volume of the extracting solution for each and then combining the two fractions obtained in a single solution.

The methylene chloride extract was dried in Rotavapor at a temperature below 35 obtaining a dark green dry residue which, resuspended in a minimum volume of methanol, represented the final extract.

The extract was stored at a temperature of -20 ° C 5 and, after having tested its activity against a test organism (Penicillium expaneum), it was used for the purification and isolation of the active ingredients.

e) Activity test

The hydrophilic and lipophilic extracts were tested for their antifungal activity (against Aspergillus flavus, Candida albicane, Colletotrichum trifolii, Fusarium roseum, Phoma sp., Penicillium expansum, Rhizoctonia solani), antibacterial (against Escherichia coli, Pseudomonus aeruginosa ) and 5 cytotoxic (against Artemia salina, Brachionus sp., HCT8 tumor line).

The hydrophilic extract showed no activity against any of the tested organisms.

The lipophilic extract completely inhibited the growth of Colletotrichum trifolii, Penicillium expansum and Rhizoctonia solani, while minor degrees of inhibition were observed against Candida albicane, Phoma sp. and Fusarium roseum.

The lipophilic extract proved lethal for both Artemia salina and Brachionus sp. and inhibited the growth of HCT8 tumor line cells in proportion to the concentrations tested.

No activity was detected against the bacterial strains tested.

e) Chemical characterization of the extract

Semi-preparative HPLC / DAD analysis

A semi-preparative HPLC column was used in order to obtain a good separation of the various components of the extract and to have a sufficient quantity of active ingredient to conduct further analyzes.

The HPLC analyzes were carried out using a liquid chromatograph (Beckman programmatale solvent module 126), equipped with a photodiode detector (Beckman diode array detector module 168) and managed by a Personal Computer through the special System Gold software (Beckman).

An 8 mm x 25 cm Kromasil 5C18 (Technicol) column was used, preceded by a 4.6 mm x 4.5 cm Ultrasphere ODS 5pm (Beckman) pre-column, with the same stationary phase and 100 μΐ loop.

The eluents used in this first phase were H20 and MeOH while the washing of the column was carried out with 100% MeOH. The elution method was one step linear gradient, with constant flow at 1 mi min <'1> (Tab. 2).

The detection of the peaks was carried out through two reading channels at 210 ± 4 and 410 ± 4 nm, and UV-vis spectra were acquired in the area between 190 and 600 nm, at the crest of the peaks.

This method allowed to isolate and accumulate a fraction from 19 to 20 minutes, containing the active principle.

Separative HPLC / DAD analysis

This analysis was carried out to have a greater resolution of the peak of the active substance, in order to determine its molecular weight with an MS-MS analysis.

A Macherey-Nagel column, CC 2S0 / 4 Nucleosil 100-5 C18 AB, 4 x 250 mm, with pre-column having the same stationary phase, 1 cm long, was used. A 20 μΐ loop was used.

H20 / H <+> (pH = 3 for HCOOH i, 5fc) and CHjCN were used as eluents. The elution method used was of the one-step sigmoidal type, with constant flow at 1 mi min <"1> (Tab. 3, Fig. 1).

The detection at the DAD (diode array detector) was carried out with two reading channels set at 210 ± 4 and 250 ± 4 nm. The second value was chosen to follow active substance, on the basis of the UV-vis spectra obtained with the previous fractionation, which revealed for this a λΜΧ at about 254 nm in methanol. UV-vis spectra were accumulated at the apex at peaks in the range 190-600 nm.

By applying this method it was possible to identify the active peak with a retention time of 19.7 min.

Using the DAD it was possible to obtain a UV-vis spectrum (190-600 nm) at the crest of the active peak with a first absorption maximum at 212 nm and a second, higher intensity, at 254 nm. These data were confirmed by the analysis of the pure substance, dried and resuspended in water.

HPLC / MS / MS analysis

In order to determine the molecular weight of the active substance, two types of mass spectrometry analyzes were conducted. The isolated active fraction was analyzed by direct infusion by analytical HPLC, and an HPLC / MS / MS was performed on the active fraction obtained by semi-preparative HPLC.

The molecular weight of the active substance was 712 m / z being the molecular ion [M + H] <*> 713 m / z.

Claims (13)

CLAIMS 1. A newly isolated cyanobacterial strain of the genus Nostoc, named Nostoc sp. KONA97, ATCC. 2. Cyanobacterial strain resulting from mutation, variation or recombination of the strain according to claim 1. 3. Crops, biomass and relative extracts obtained from the strain according to claims 1 or 2. 4. Bioactive molecules present in crops or biomass of Nostoc sp. KONA97 or in the corresponding extracts according to claim 3. 5. Bioactive molecules according to claim 4 with molecular weight 712. 6. Bioactive molecules according to claims 4-5, in any case obtained by isolation from microorganisms or synthetically. 7. Use in the agricultural field of crops, biomass, extracts, bioactive molecules of Nostoc sp. KONA97 according to claims 1 to 6. 8. Use of cultures, biomasses, extracts, bioactive molecules of Nostoc sp. KONA97 according to claim 7 as antifungals, antibacterials, insecticides, nematocides and antimicrobials in general. 9. Use of lipophilic extracts of Nostoc sp. K0NA97 according to claim 7 against Colletotrichum trifolii Penicillium expansuin, Rhizoctonia solani Candida. apricans, Phoma sp., Fusarium roseum, Artemia salina; Brachionus sp. 10. Use of cultures, biomasses, extracts, bioactive molecules of Nostoc sp. KONA97 according to claims 1 to 6 in the pharmaceutical field. 11. Use of cultures, biomasses, extracts, bioactive molecules of Nostoc sp. KONA97 according to claim 10 as anticancer. 12. Use of lipophilic extracts of Nostoc sp. KONA97 according to claim 11 to inhibit the growth of cells of the HCT8 tumor line. 13. Method for producing biomass of Nostoc sp. K0NA97, according to claims 1 or 2, comprising the steps of: - inoculating the maintenance culture of the cyanobacterium in liquid or agar medium; - incubation in a thermostat equipped with a stirring system, at a temperature of 30 ° C, under continuous lighting conditions and in an air-carbon dioxide atmosphere, - massive culture in closed photobioreactors and / or open systems. By proxy: UNIVERSITY OF FLORENCE; CNR NATIONAL RESEARCH COUNCIL,